Method for the diagnosis and prognosis of malignant diseases

ABSTRACT

Methods for the diagnosis of cancer by determining the neoplastic status of a cell by probing the cell plasma membrane for the presence of nucleolin are provided, as are kits to carry out such tests.

[0001] The invention is based on the discovery of a correlation betweennucleolin cellular plasma membrane expression and the presence andaggressiveness of neoplastic cells. Thus, the invention is drawn to thedetection and prognosis of pre-malignant conditions and malignantdiseases and disorders and kits for detection and prognosis.

BACKGROUND

[0002] Being well-prepared for battle engenders success; when the foe iscancer, early detection results in a greater likelihood that medicalintervention will be successful. At early stages, treatments can oftenbe targeted only to the affected tissues, diminishing side effects. Ifnot caught early, cancer cells may metastasize and spread throughout thebody. The prognosis in this case is more dire, and medical treatmentsare often applied systemically, killing not only cancer cells, but largenumbers of healthy cells.

[0003] A hallmark of a cancer cell is uncontrolled proliferation. Cancercells may also exhibit morphological and functional aberrations. Cancercells may display less organized cellular morphology; for example,losing the asymmetric organelle and structural organization (cellpolarity) that allows for proper cell function. Cell-cell andcell-substratum contacts, the specificities of which are also necessaryfor normal function, are often modulated or lost. Functionally, thecells may carry on few, if any, wild-type functions, or may haveexaggerated, unregulated normal functions, such as hormone secretion.Such cells regress to early developmental stages, appearing lessdifferentiated than their wild-type (i.e., normal) parents.

[0004] Cancer cells also often mis-express or mis-target proteins toinappropriate cellular compartments. Proteins may be up- ordown-regulated; even proteins not usually expressed by a specific celltype can be expressed by the transformed counterpart. Proteinmis-expression can have a plethora of downstream cellular effects,including drastic changes in membrane composition, organelle formation,or physiology. Mis-targeting of proteins (and other molecules, such aslipids, etc.) also contributes to the loss of cell polarity.

[0005] Detecting cancer growths are complicated by a variety of factors,including protein mis-expression and cellular dedifferentiation. Forexample, cancers can proceed gradually, being present in only smallnumbers that are difficult to detect. A common diagnostic approach isthe use of certain proteins as “markers” to distinguish cancer cellsfrom healthy ones. For example, prostate cancer diagnoses often useprostate-specific antigen (PSA). These assays require that a marker bedefined, have detection agents, and be readily available. However, amarker that is present in a many, if not most, cancers has yet to bedefined. Such a marker would remarkably facilitate diagnosis.

SUMMARY

[0006] In a first aspect, the invention is drawn to methods ofdetermining a neoplastic state of a cell, comprising detecting thepresence of nucleolin on the plasma membrane of a cell, includingmammalian cells, such as human; the cell may also be lysed. Plasmamembrane nucleolin is detected by using an anti-nucleolin agent, such asanti-nucleolin antibodies or oligonucleotides, including those ofsequences of SEQ ID NOs:1-7; 9-17; 19-31.

[0007] In a second aspect, the invention is drawn to methods ofdetermining a phenotype of a neoplastic cell, comprising quantifying theamount of plasma membrane nucleolin of the cell, including mammaliancells, such as human; the cell may also be lysed. Plasma membranenucleolin is detected by using an anti-nucleolin agent, such asanti-nucleolin antibodies or oligonucleotides, including those ofsequences of SEQ ID NOs:1-7; 9-17; 19-31.

[0008] In another aspect, the invention is drawn to kits for determininga neoplastic state of a cell, comprising an anti-nucleolin agent and acontrol sample. Such kits may also contain reagents that bind tonucleolin, reagent-nucleolin complexes and other components, such as afixative, a buffer, plasticware, serum, serum albumin, non-fat milk,membranes and instructions.

[0009] In another aspect, the invention pertains to methods of detectingsmall lung cell carcinoma in a subject, comprising collecting a samplecontaining lung cells from a subject; and detecting the presence ofplasma membrane nucleolin in the cells. Samples include sputum; subjectsmay be human.

[0010] In yet another aspect, the invention is drawn to methods ofdiagnosing tumor, pre-malignant or malignant cells, comprisingcollecting a sample from a subject comprising cells, sending the sampleto a testing center, determining the neoplastic state of the cells byprobing for cell plasma membrane nucleolin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011]FIG. 1 shows nuclear nucleolin staining in various cell lines.Shown are immunofluorescent (B, D, F, H) and parallel phase contrastmicrographs (A, C, E, G). Cell lines that were analyzed were: DU145prostate cancer cells (A, B), MDA-MB-231 breast cancer cells (C, D),HeLa cervical cancer cells (E, F) and HS27 normal skin cells (G, H). Ananti-nucleolin antibody was used; the cells were permeabilized beforestaining to allow the antibody access to the cytoplasmic and nuclearcompartments.

[0012]FIG. 2 shows plasma membrane nucleolin staining in the cell linesshown in FIG. 1. Shown are immunofluorescent (B, D, F, H) and parallelphase contrast micrographs (A, C, E, G). Cell lines that were analyzedwere: DU145 prostate cancer cells (A, B), MDA-MB-231 breast cancer cells(C, D), HeLa cervical cancer cells (E, F) and HS27 normal skin cells (G,H). An anti-nucleolin antibody was used; the cells were notpermeabilized before staining, allowing the antibody access to only theplasma membrane.

[0013]FIG. 3 shows the comparative proliferation rates of cell lines asmeasured by MTT assay. Square, DU145; diamonds, HeLa; circles, HS27.Although MDA-MB-231 was not included in this experiment, proliferationrates for these four cell lines have been determined to beDU145>MDA-MB-231>HeLa >HS27. Note that the cell lines with high levelsof plasma membrane nucleolin correspond to those with the most rapidproliferation (DU145 and HeLa; see FIG. 2).

[0014]FIG. 4 shows phase contrast (B, D) and immunofluorescent image (A,C) of a paraffin-embedded specimen resected from a patient with squamouscell carcinoma of the head and neck. The specimen was stained for plasmamembrane nucleolin and counter-stained with propidium iodide to showcell nuclei. Images (C) and (D) show images (A) and (B) overlaid withmarkings to better show nucleolin staining. The area 1 encompassed bythe white line includes intense nucleolin staining, while the areasoutside of 1 show little 3 or no 2 signal.

[0015]FIG. 5 shows phase contrast (B, D) and immunofluorescent images(A, C) of small cell (NCI-H82) and non-small cell lung (NCI-H1299)cancer cell lines placed onto a microscope slide using a cytospinner.Samples were stained for plasma membrane nucleolin and counter-stainedwith propidium iodide to show cell nuclei. Cells with exceptionallywell-stained plasma membranes are denoted by asterisks (*).

[0016]FIG. 6 shows phase contrast (B, D, F) and immunofluorescent images(A, C, E) of peripheral blood (A, B) or bone marrow (C, D and E, F) fromhuman subjects. Samples were stained for plasma membrane nucleolin andcounter-stained with propidium iodide to show cell nuclei. Highlystained cells for nucleolin are marked with an asterisk (*); these wereonly seen in those patients suffering from carcinomas (A, B and C, D),while cells from a healthy patient did not display any plasma membranenucleolin staining (E, F).

DETAILED DESCRIPTION

[0017] The invention is based on the discovery of a correlation betweennucleolin plasma membrane expression and the presence and aggressivenessof neoplastic cells.

[0018] The invention provides compositions and methods for the detectionof neoplastic cells, such as malignant and pre-malignant cells, using aneoplastic antigen that is broadly expressed, nucleolin. However, theexpression of nucleolin is insufficient to indicate malignancy(nucleolin is found in every nucleated cell); but the localization ofnucleolin on the cell surface is indicative. While increased amounts ofnuclear nucleolin, as visualized by silver staining, has been used as adiagnostic tool for cancer, the unexpected discovery that nucleolinfound on the cell surface correlates with a pre-malignant or malignantphenotype can facilitate cancer diagnosis and prognosis.

[0019] The advantages of using surface-localized nucleolin include:

[0020] (1) Improved accuracy. Assaying for plasma membrane nucleolinmore accurately identifies the malignant phenotype compared tosilver-staining for nuclear nucleolin.

[0021] (2) Specificity. Plasma membrane nucleolin is not normallyobserved in the plasma membrane of most wild-type (healthy) cells. Thus,unlike many other diagnostic tests that require analyzing the amount ofa marker, plasma membrane nucleolin detection can be used qualitatively.A more fail-proof “yes-no” type of test is therefore feasible. Thisaspect also allows for testing on both single cells and tissue samples.

[0022] (3) Broad applicability. Many different cancers may be detectedby examining for plasma membrane nucleolin.

[0023] (4) Multifunctional. The methods are prognostic as well asdiagnostic: the rate of cellular proliferation typically correlates withlevels of plasma membrane nucleolin.

[0024] While investigating the anti-proliferative activity ofnon-antisense guanosine-rich oligonucleotides (GROs) on cancer cells, itwas found that such anti-proliferative GROs bind nucleolin to exerttheir effects (Bates et al., 1999; Miller et al., WO 00/61597, 2000).Nucleolin (Bandman et al., U.S. Pat. No. 5,932,475, 1999) is anabundant, non-ribosomal protein of the nucleolus, the site of ribosomalgene transcription and packaging of pre-ribosomal RNA. This 707 aminoacid phosphoprotein has a multi-domain structure consisting of ahistone-like N-terminus, a central domain containing four RNArecognition motifs and a glycine/arginine-rich C-terminus and has anapparent molecular weight of 110 kD. Its multiple domain structurereflects the remarkably diverse functions of this multifaceted protein(Ginisty et al., 1999; Srivastava and Pollard, 1999; Tuteja and Tuteja,1998). Nucleolin has been implicated in many fundamental aspects of cellsurvival and proliferation. Most understood is the role of nucleolin inribosome biogenesis. Other functions may include nucleocytoplasmictransport, cytokinesis, nucleogenesis and apoptosis.

[0025] Nucleolin synthesis has been correlated with increased rates ofcell division (cell proliferation); nucleolin levels are thereforehigher in tumor cells compared to most normal cells (Tuteja and Tuteja,1998). Nucleolin is one of the nuclear organizer region (NOR) proteinswhose levels, as measured by silver staining, are assessed bypathologists as a marker of cell proliferation and an indicator ofmalignancy (Derenzini, 2000).

[0026] Also present in the cell plasma membrane in a few cell types,such as lymphocytes and inner medullary collecting duct cells, nucleolinhas been hypothesized to function as a receptor (e.g., Callebaut et al.,1998; Sorokina and Kleinman, 1999). However, the role of plasma membranenucleolin is not well understood. In addition, it is not clear whetherthe plasma membrane nucleolin is identical to the nucleolar protein, orif it represents a different isoform or nucleolin-like protein. However,the expression of plasma membrane nucleolin is specific to neoplasticcells (such as malignant or pre-malignant); thus the function of plasmamembrane nucleolin need not be known for diagnostic and prognosticpurposes.

[0027] Definitions

[0028] Neoplasm, Malignancy, Tumor, Cancer Cells

[0029] A neoplasm is an abnormal tissue growth resulting from neoplasticcells, cells that proliferate more rapidly and uncontrollably thannormal cells. Usually partially or completely structurally disorganized,neoplasms lack functional coordination with the corresponding normaltissue. Neoplasms usually form a distinct tissue mass that may be eitherbenign (tumor) or malignant (cancer).

[0030] Cancer cells invade surrounding tissues, may metastasize todistant sites, and are likely to recur after attempted removal, causingdeath of a subject if not adequately treated. In addition to structuraldisorganization, cancer cells usually regress to more primitive orundifferentiated states (anaplasia), although morphologically andbiochemically, they may still exhibit many functions of thecorresponding wild-type cells. Carcinomas are cancers derived fromepithelia;

[0031] sarcomas are derived from connective tissues.

[0032] Cancers may be more aggressive or less aggressive. The aggressivephenotype of a cancer cell refers to the proliferation rate and theability to form tumors and metastasize in nude mice. Aggressive cancersproliferate more quickly, more easily form tumors and metastasize thanless-aggressive tumors.

[0033] Neoplastic State

[0034] The term “neoplastic state” refers to three conditions: normal,pre-malignant and malignant. “Normal” refers to a growth or cell that isclinically normal (healthy). “Pre-malignant” refers to a growth or cellthat is on the pathway to malignancy, but at the time of examination,would not be classified as malignant by conventional methods.“Malignant” refers to a cell or growth that has at least one of thefollowing properties: locally invasive, destructive growth andmetastasis.

[0035] GROs and Other Polypeptide-Binding Oligonucleotides

[0036] Oligonucleotides are available that specifically bind topolypeptides, such as nucleolins. Examples of such are GROs, which areguanosine-rich oligonucleotides. Characteristics of GROs include:

[0037] (1) having at least 1 GGT motif

[0038] (2) preferably having 4-100 nucleotides, although GROs havingmany more nucleotides are possible

[0039] (3) having chemical modifications to improve stability.

[0040] Especially useful GROs form G-quartet structures, as indicated bya reversible thermal denaturation/renaturation profile at 295 nm (Bateset al., 1999). Preferred GROs also compete with a telomereoligonucleotide for binding to a target cellular protein in anelectrophoretic mobility shift assay (Bates et al., 1999).

[0041] Other oligonucleotides may have high binding specificity fornucleolin.

[0042] Anti-Nucleolin Agent

[0043] An “anti-nucleolin agent” binds to nucleolin. Examples includeanti-nucleolin antibodies and certain oligonucleotides.

[0044] Embodiments

[0045] The following embodiments are given as examples of various waysto practice the invention. Many different ways of practicing theinvention are also possible.

[0046] In all embodiments, the underlying principle is to specificallydifferentiate between plasma membrane nucleolin and nuclear nucleolin.Plasma membrane nucleolin, as discovered by the applicants, correlateswith cells that are in a neoplastic state; furthermore, the amount ofplasma membrane nucleolin also indicates the aggressiveness of thesecells; that is, the higher the plasma membrane nucleolin expression, themore aggressive the cells. Various techniques allow a user todifferentiate between nuclear and plasma membrane nucleolin. Detectiontechniques, wherein the nucleolin-detecting reagents have exclusiveaccess to extracellular portions of the cell (and consequentlycell-plasma membrane nucleolin), or biochemical techniques, whereineither the surface plasma membrane and/or surface proteins are separatedfrom other cellular components and compartments, are also useful.

[0047] In an embodiment, nucleolin is detected directly on the cellsurface. A cell is isolated from a subject and plasma membrane nucleolindetected using an agent that binds nucleolin. Cells may be isolated byany known technique. An isolated cell may comprise a larger tissuesample containing cells that are not neoplastic. Detection proceduresuse anti-nucleolin antibodies that bind extracellular nucleolinepitopes; these antibodies may be directly labeled or when bound,detected indirectly. Other useful plasma membrane nucleolin detectionagents include GROs that specifically bind nucleolin. Useful procedures,such as fluorescence-activated cell sorting (FACS) orimmunofluorescence, employ fluorescent labels, while other cytologicaltechniques, such as histochemical, immunohistochemical and othermicroscopic (electron microscopy (EM), immuno-EM) techniques use variousother labels, either colorimetric or radioactive. The various reagentsmay be assembled into kits.

[0048] In another embodiment, cells are isolated from a subject andextracted. Plasma plasma membranes and/or proteins are then isolated(such as via differential extraction, or differential physical celldisruption, differential centrifugation of detergent-extracted cells,etc.), and then nucleolin detected in the isolated membranes using anagent that binds nucleolin. In general, useful techniques to detectnucleolin include those wherein the extract is placed on a substrate,and the substrate probed with a nucleolin-detecting reagent. Examples ofsuch techniques include polypeptide dot blots and Western blots,biochips, protein arrays, etc. Other detection formats includeenzyme-linked immunosorbent assays (ELISAs) in their manifoldmanifestations (Ausubel et al., 1987). In embodiments wherein plasmamembrane surface molecules are physically separated from most of theother cellular components and compartments, the nucleolin-binding agentsneed not specifically recognize any extracellular portions of nucleolin.The various reagents may be assembled into kits.

[0049] In a further embodiment, the methods of the invention aredirected to detecting lung cancer, such as lung small cell carcinomas.Plasma membrane nucleolin expression is useful for detection andprognosis.

[0050] In yet another embodiment, methods of treating cells in aneoplastic state including cancer and tumor cells are provided,exploiting plasma membrane nucleolin that acts as a beacon.Administration of anti-nucleolin antibodies, which may be conjugated toa toxin or other means of stimulating cell death or incurring cellnecrosis, results in the removal of plasma membrane nucleolin-expressingcells.

[0051] Practicing the Invention

[0052] The methods of the invention include in part the steps ofcollecting cells from a subject and detecting plasma membrane nucleolin.The following, not meant to limit the invention, is presented to aid thepractitioner in carrying out the invention, although other methods,techniques, cells, reagents and approaches can be used to achieve theinvention.

[0053] Cells

[0054] Cells or tissue samples are collected from a subject. The subjectis a vertebrate, more preferably a mammal, such as a monkey, dog, cat,rabbit, cow, pig, goat, sheep, horse, rat, mouse, guinea pig, etc.; andmost preferably a human. Any technique to collect the desired cells maybe employed, including biopsy, surgery, scrape (inner cheek, skin, etc.)and blood withdrawal. Any appropriate tool may be used to carry out suchtasks. It is not necessary to isolate the test population (i.e., thosecells being tested for neoplastic state) from those cells and tissues(contaminating material) that are not being tested, except in some casesusing biochemical methods that include extraction. In this last case,the test population need not be completely isolated from contaminatingmaterials, but should either predominate or be easily distinguishable(e.g., morphologically (structurally, specific markers) orbiochemically).

[0055] For those methods that analyze lung carcinomas, sputum collectionis an attractive and easily obtained sample. The term “sputum” as usedherein refers to expectorated matter made up of saliva and dischargesfrom the respiratory airways. Sputum is a highly complex material thathas a pronounced gel-like structure.

[0056] For collection of sputum, Byrne, et al., (Byrne, 1986) suggestthat the patient collect material, raised by several deep coughs, in acontainer with a lid. Alternatively, sputum can be collected by using abronchoscope (Kim et al., 1982). Specific devices or agents may be usedto facilitate sputum collection (Babkes et al., U.S. Pat. No. 6,325,785,2001; King and Speert, U.S. Pat. No. 6,339,075, 2002; Rubin andNewhouse, U.S. Pat. No. 5,925,334, 1999). Other methods of sputumcollection are also available.

[0057] Cell Culture

[0058] In some cases, culturing the harvested cells is desirable toaugment their numbers so that plasma membrane nucleolin detection isfacilitated. Suitable media and conditions for generating primarycultures are well known. The selection of the media and cultureconditions vary depending on cell type and may be empiricallydetermined. For example, skeletal muscle, bone, neurons, skin, liver,and embryonic stem cells are grown in media that differs in theirspecific contents. Furthermore, media for one cell type may differsignificantly from laboratory to laboratory and institution toinstitution. To keep cells dividing, serum, such as fetal calf serum(FCS) (also known as fetal bovine serum (FBS)), is added to the mediumin relatively large quantities, 5%-30% by volume, depending on cell ortissue type. Other sera include newborn calf serum (NCS), bovine calfserum (BCS), adult bovine serum (ABS), horse serum (HS), human, chicken,goat, porcine, rabbit and sheep sera. Serum replacements may also beused, such as controlled process serum replacement-type (CPSR; 1 or 3)or bovine embryonic fluid. Specific purified growth factors or cocktailsof multiple growth factors can also be added or sometimes substitutedfor serum. Specific factors or hormones that promote proliferation orcell survival can also be used.

[0059] Examples of suitable culture media include Iscove's ModifiedDulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM),Minimal Essential Medium Eagle (MEM), Basal Medium Eagle (BME), Click'sMedium, L-15 Medium Leibovitz, McCoy's 5A Medium, Glasgow MinimumEssential Medium (GMEM), NCTC 109 Medium, Williams' Medium E, RPMI-1640,and Medium 199. A medium specifically developed for a particular celltype/line or cell function, e.g. Madin-Darby Bovine Kidney GrowthMedium, Madin-Darby Bovine Kidney Maintenance Medium, various hybridomamedia, Endothelial Basal Medium, Fibroblast Basal Medium, KeratinocyteBasal Medium, and Melanocyte Basal Medium are also known. If desired, aprotein-reduced or -free and/or serum-free medium and/or chemicallydefined, animal component-free medium may be used, e.g., CHO, GeneTherapy Medium or QBSF Serum-free Medium (Sigma Chemical Co.; St. Louis,Mo.), DMEM Nutrient Mixture F-12 Ham, MCDB (105, 110, 131, 151, 153, 201and 302), NCTC 135, Ultra DOMA PF or HL-1 (both from Biowhittaker;Walkersville, Md.), may be used.

[0060] The medium can be supplemented with a variety of growth factors,cytokines, serum, etc., depending on the cells being cultured. Examplesof suitable growth factors include: basic fibroblast growth factor(bFGF), vascular endothelial growth factor (VEGF), epidermal growthfactor (EGF), transforming growth factors (TGFα and TGFβ), plateletderived growth factors (PDGFs), hepatocyte growth factor (HGF),insulin-like growth factor (IGF), insulin, erythropoietin (EPO), andcolony stimulating factor (CSF). Examples of suitable hormone additivesare estrogen, progesterone, testosterone or glucocorticoids such asdexamethasone. Examples of cytokine medium additives are interferons,interleukins or tumor necrosis factor-α (TNFα). Salt solutions may alsobe added to the media, including Alseverr's Solution, Dulbecco'sPhosphate Buffered Saline (DPBS), Earle's Balanced Salt Solution, Gey'sBalanced Salt Solution (GBSS), Hanks' Balanced Salt Solution (HBSS),Puck's Saline A, and Tyrode's Salt Solution. If necessary, additives andculture components in different culture conditions be can optimized, asthese may alter cell response, activity lifetime or other featuresaffecting bioactivity. In addition, the surface on which the cells aregrown can be coated with a variety of substrates that contribute tosurvival, growth and/or differentiation of the cells. These substratesinclude but are not limited to, laminin, EHS-matrix, collagens,poly-L-lysine, poly-D-lysine, polyomithine and fibronectin. Whenthree-dimensional cultures are desired, extracellular matrix gels may beused, such as collagen, EHS-matrix, or gelatin (denatured collagen).Cells may be grown on top of such matrices, or may be cast within thegels themselves.

[0061] If desired, the media may be further supplemented with reagentsthat limit acidosis of the cultures, such as buffer addition to themedium (such as N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid(BES), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris),N-(20hydroxyethyl)piperazine-N′3-propanesulfonic acid (EPPS or HEPPS),glyclclycine, N-2-hydroxyehtylpiperazine-N′-2-ethanesulfonic acid(HEPES), 3-(N-morpholino)propane sulfonic acid (MOPS),Piperazine-N,N′-bis(2-ethane-sulfonic acid) (PIPES), sodium bicarbonate,3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid)TAPSO, (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),N-tris(hydroxymethyl)methyl-glycine (Tricine),tris(hydroxymethyl)-aminomethane (Tris), etc.). Frequent medium changesand changes in the supplied CO₂ (often approximately 5%) concentrationmay also be used to control acidosis.

[0062] Gases for culture typically are about 5% carbon dioxide and theremainder nitrogen, but optionally may contain varying mounts of nitricoxide (starting as low as 3 ppm), carbon monoxide and other gases, bothinert and biologically active. Carbon dioxide concentrations typicallyrange around 5%, but may vary between 2-10%. Both nitric oxide andcarbon monoxide, when necessary, are typically administered in verysmall amounts (i.e. in the ppm range), determined empirically or fromthe literature. The temperature at which the cells will grow optimallycan be empirically determined, although the culture temperature willusually be within the normal physiological range of the animal fromwhich the cells were isolated.

[0063] Detecting Nucleolin: Antibody-Based Methods

[0064] Nucleolin can be detected at the protein level in cells, tissuesections, cultured cells and extracts thereof. Immunochemical methods todetect protein expression are well known and include, but are notlimited to, Western blotting, immunoaffinity purification,immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), dot orslot blotting, radioimmunoassay (RIA), immunohistochemical detection,immunocytochemical staining, and flow cytometry. Common procedures andinstructions using antibodies have been well addressed (e.g., Harlow andLane, 1988; Harlow and Lane, 1999). Selected antibodies that are usefulfor detecting plasma membrane nucleolin are shown in Table 1. TABLE 1Anti-nucleolin antibodies Antigen Antibody Source source Notes p7-1A4mouse Developmental Xenopus laevis IgG₁ monoclonal antibody StudiesHybridoma oocytes (mAb) Bank (University of Iowa; Ames, IA) sc-8031mouse mAb Santa Cruz Biotech human IgG₁ (Santa Cruz, CA) sc-9893 goatSanta Cruz Biotech human IgG polyclonal Ab (pAb) sc-9892 goat pAb SantaCruz Biotech human IgG clone 4E2 mouse MBL International human IgG₁ mAb(Watertown, MA) clone 3G4B2 mouse Upstate dog (MDCK IgG_(1k) mAbBiotechnology (Lake cells) Placid, NY)

[0065] If additional anti-plasma membrane nucleolin antibodies aredesired, they can be produced using well-known methods (Harlow and Lane,1988; Harlow and Lane, 1999). For example, (pAbs) can be raised in amammalian host by one or more injections of an immunogen, such as anextracellular domain of surface-expressed nucleolin, and, if desired, anadjuvant. Typically, the immunogen (and adjuvant) is injected in amammal by a subcutaneous or intraperitoneal injection. The immunogen mayinclude components such as polypeptides (isolated, non-isolated, orrecombinantly produced), cells or cell fractions. Examples of adjuvantsinclude Freund's complete and monophosphoryl Lipid A synthetic-trehalosedicorynomycolate (MPL-TDM). To improve the immune response, an immunogenmay be conjugated to a polypeptide that is immunogenic in the host, suchas keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulinor soybean trypsin inhibitor. Alternatively, pAbs may be made inchickens, producing IgY molecules (Schade et. al., 1996).

[0066] mAbs may also be made by immunizing a host or lymphocytes from ahost, harvesting the mAb-secreting (or potentially secreting)lymphocytes, fusing those lymphocytes to immortalized cells (e.g.,myeloma cells), and selecting those cells that secrete the desired mAb(Goding, 1996). If desired, the mAbs may be purified from the culturemedium or ascites fluid by conventional procedures such as proteinA-sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, ammonium sulfate precipitation or affinity chromatography(Harlow and Lane, 1988; Harlow and Lane, 1999).

[0067] The antibodies may be whole antibodies and fragments orderivatives thereof. For example, when assaying live cells, using F_(ab)fragments will eliminate cross-linking, thus preventing the cells fromendocytosing the bound antibodies.

[0068] An approach using antibodies to detect the presence of an antigenwill include one or more, if not all, of the following steps:

[0069] (1) Preparing the entity being tested for plasma membranenucleolin by washing with buffer or water

[0070] (2) Blocking non-specific antibody binding sites

[0071] (3) Applying the antibody (e.g., nucleolin)

[0072] (4) Detecting bound antibody, either via a detectablelabeled-secondary antibody that recognizes the primary antibody or adetectable label that has been directly attached to, or associated with,the bound (anti-nucleolin) antibody.

[0073] Substrates may be washed with any solution that does notinterfere with the epitope structure. Common buffers include saline andbiological buffers, such as bicine, tricine, and Tris.

[0074] Non-specific binding sites are blocked by applying a proteinsolution, such as bovine serum albumin (BSA; denatured or native), milkproteins, or in the cases wherein the detecting reagent is a secondaryantibody, normal serum or immunoglobulins from a non-immunized hostanimal whose species is the same origin as the detecting antibody. Forexample, a procedure using a secondary antibody made in goats wouldemploy normal goat serum (NGS).

[0075] The substrate is then reacted with the antibody of interest. Theantibody may be applied in any form, such as F_(ab) fragments andderivatives thereof, purified antibody (by affinity, precipitation,etc.), supernatant from hybridoma cultures, ascites, serum orrecombinant antibodies expressed in recombinant cells. The antibody maybe diluted in buffer or media, often with a protein carrier such as thesolution used to block non-specific binding sites; the useful antibodyconcentration is usually determined empirically. In general, polyclonalsera, purified antibodies and ascites may be diluted 1:50 to 1:200,000,more often, 1:200 to 1:500. Hybridoma supernatants may be diluted 1:0 to1:10, or may be concentrated by dialysis or ammonium sulfateprecipitation (or any other method that retains the antibodies ofinterest but at least partially removes the liquid component andpreferably other small molecules, such as salts) and diluted ifnecessary. Incubation with antibodies may be carried out for as littleas 20 minutes at 37° C., 2 to 6 hours at room temperature (approximately22° C.), or 8 hours or more at 4° C.

[0076] To detect an antibody-antigen complex, a label may be used. Thelabel may be coupled to the binding antibody, or to a second antibodythat recognizes the first antibody, and is incubated with the sampleafter the primary antibody incubation and thorough washing. Suitablelabels include fluorescent moieties, such as fluorescein isothiocyanate;fluorescein dichlorotriazine and fluorinated analogs of fluorescein;naphthofluorescein carboxylic acid and its succinimidyl ester;carboxyrhodamine 6G; pyridyloxazole derivatives; Cy2, 3 and 5;phycoerythrin; fluorescent species of succinimidyl esters, carboxylicacids, isothiocyanates, sulfonyl chlorides, and dansyl chlorides,including propionic acid succinimidyl esters, and pentanoic acidsuccinimidyl esters; succinimidyl esters of carboxytetramethylrhodamine;rhodamine Red-X succinimidyl ester; Texas Red sulfonyl chloride; TexasRed-X succinimidyl ester; Texas Red-X sodium tetrafluorophenol ester;Red-X; Texas Red dyes; tetramethylrhodamine; lissamine rhodamine B;tetramethylrhodamine; tetramethylrhodamine isothiocyanate;naphthofluoresceins; coumarin derivatives; pyrenes; pyridyloxazolederivatives; dapoxyl dyes; Cascade Blue and Yellow dyes; benzofuranisothiocyanates; sodium tetrafluorophenols;4,4-difluoro-4-bora-3a,4a-diaza-s-indacene. Suitable labels furtherinclude enzymatic moieties, such as alkaline phosphatase or horseradishperoxidase; radioactive moieties, including ³⁵S and ¹³⁵I-labels; avidin(or streptavidin)-biotin-based detection systems (often coupled withenzymatic or gold signal systems); and gold particles. In the case ofenzymatic-based detection systems, the enzyme is reacted with anappropriate substrate, such as 3, 3′-diaminobenzidine (DAB) forhorseradish peroxidase; preferably, the reaction products are insoluble.Gold-labeled samples, if not prepared for ultrastructural analyses, maybe chemically reacted to enhance the gold signal; this approach isespecially desirable for light microscopy. The choice of the labeldepends on the application, the desired resolution and the desiredobservation methods. For fluorescent labels, the fluorophore is excitedwith the appropriate wavelength and the sample observed using amicroscope, confocal microscope, or FACS machine. In the case ofradioactive labeling, the samples are contacted with autoradiographyfilm, and the film developed; alternatively, autoradiography may also beaccomplished using ultrastructural approaches. Alternatively,radioactivity may be quantified using a scintillation counter.

[0077] Cytological-Based Approaches:

[0078] Immunofluorescence/Immunohistochemical

[0079] Protein expression by cells or tissue can be ascertained byimmunolocalization of an antigen. Generally, cells or tissue arepreserved by fixation, exposed to an antibody that recognizes theepitope of interest, such as a nucleolin, and the bound antibodyvisualized.

[0080] Any cell, cell line, tissue, or even an entire organism isappropriate for fixation. Cells may be cultured in vitro as primarycultures, cell lines, or harvested from tissue and separatedmechanically or enzymatically. Tissue may be from any organ, plant oranimal, and may be harvested after or prior to fixation. Fixation, ifdesired, may be by any known means; the requirements are that theprotein to be detected be not rendered unrecognizable by the bindingagent, most often an antibody. Appropriate fixatives includeparaformaldehyde-lysine-periodate, formalin, paraformaldehyde, methanol,acetic acid-methanol, glutaraldehyde, acetone, Kamovsky's fixative, etc.The choice of fixative depends on variables such as the protein ofinterest, the properties of a particular detecting reagent (such as anantibody), the method of detection (fluorescence, enzymatic) and themethod of observation (epifluorescence microscopy, confocal microscopy,light microscopy, electron microscopy, etc.). The sample is usuallyfirst washed, most often with a biological buffer, prior to fixation.Fixatives are prepared in solution or in biological buffers; manyfixatives are prepared immediately prior to applying to the sample.Suitable biological buffers include saline (e.g., phosphate bufferedsaline), N-(carbamoylmethyl)-2-aminoethanesulfonic acid (ACES),N-2-acetamido-2-iminodiacetic acid (ADA), bicine, bis-tris,3-cyclohexylamino-2-hydroxy-1-propanesulfonic acid (CAPSO),ethanolamines, glycine, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonicacid (HEPES), 2-N-morpholinoethanesulfonic acid (MES),3-N-morpholinopropanesulfonic acid (MOPS),3-N-morpholino-2-hyrdoxy-propanesulfonic acid (MOPSO),piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), tricine,triethanolamine, etc. An appropriate buffer is selected according to thesample being analyzed, appropriate pH, and the requirements of thedetection method. A useful buffer is phosphate buffered saline (PBS).After fixation, the sample may be stored in fixative, preferably fresh,or temporarily or indefinitely, at a temperature between about 4° C. toabout 22° C.

[0081] After fixation from 5 minutes to 1 week, depending on the samplesize, sample thickness, and viscosity of the fixative, the sample iswashed in buffer. If the sample is thick or sections are desired, thesample may be embedded in a suitable matrix. For cryosectioning, sucroseis infused, and embedded in a matrix, such as OCT Tissue Tek (AndwinScientific; Canoga Park, Calif.) or gelatin. Samples may also beembedded in paraffin wax, or resins suitable for electron microscopy,such as epoxy-based (Araldite, Polybed 812, Durcupan ACM, Quetol,Spurr's, or mixtures thereof; Polysciences, Warrington, Pa.), acrylates(London Resins (LR White, LR gold), Lowicryls, Unicryl; Polysciences),methylacrylates (JB-4, OsteoBed; Polysciences), melamine (Nanoplast;Polysciences) and other media, such as DGD, Immuno-Bed (Polysciences)and then polymerized. Resins that are especially appropriate includehydrophilic (such as Lowicryls, London Resins, water-soluble Durcupan,etc.) since these are less likely to denature the protein of interestduring polymerization and will not repel antibody solutions. Whenembedded in wax or resin, samples are dehydrated by passing them througha concentration series of ethanol or methanol; in some cases, othersolvents may be used, such as polypropylene oxide. Embedding may occurafter the sample has been reacted with the detecting agents, or samplesmay be first embedded, sectioned (via microtome, cyrotome, orultramicrotome), and then the sections reacted with the detectingreagents. In some cases, the embedding material may be partially orcompletely removed before detection to facilitate antigen access.

[0082] In some instances, the nucleolin epitope(s) to which the antibodybinds may be rendered unavailable because of fixation. Antigen retrievalmethods can be used to make the antigen available for antibody binding.Many recourses are available (reviewed in, for example, McNicol andRichmond, 1998; Robinson and Vandre, 2001; Shi et al., 2001). Commonmethods include using heat supplied from autoclaves, microwaves, hotwater or buffers, pressure cookers, or other sources of heat. Often thesources of heat are used in sequence; the samples must often be insolution (e.g., microwave treatments). Detergent treatment may alsounmask antigens, such as sodium dodecyl sulfate (SDS, 0.25% to 1%) orother denaturing detergents. Chemical methods include strong alkalis(such as NaOH), prolonged immersion in water, urea, formic acid andrefixation in zinc sulfate-formalin. In other instances, proteolyticenzyme treatment will modify the antigen such that it is available tothe antibody. Any number of proteases may be used, such as trypsin.These methods may be combined to achieve optimal results. The choice ofthe antigen retrieval method will depend on the sample, its embedment(if any), and the anti-nucleolin antibody.

[0083] Especially in the cases of immunofluorescent or enzymaticproduct-based detection, background signal due to residual fixative,protein cross-linking, protein precipitation or endogenous enzymes maybe quenched, using, e.g., ammonium chloride or sodium borohydride or asubstance to deactivate or deplete confounding endogenous enzymes, suchas hydrogen peroxide which acts on peroxidases. To detect intracellularproteins in samples that are not to be sectioned, samples may bepermeabilized. Permeabilizing agents include detergents, such ast-octylphenoxypolyethoxyethanols, polyoxyethylenesorbitans, and otheragents, such as lysins, proteases, etc.

[0084] Non-specific binding sites are blocked by applying a proteinsolution, such as bovine serum albumin (BSA; denatured or native), milkproteins, or preferably in the cases wherein the detecting reagent is anantibody, normal serum or IgG from a non-immunized host animal whosespecies is the same is the same origin of the detecting antibody.

[0085] Flow Cytometry/Fluorescence-Activated Cell Sorting (FACS)

[0086] Methods of performing flow cytometry are well known (Orfao andRuiz-Arguelles, 1996). Because plasma membrane nucleolin is beingprobed, cell permeabilization that allows access to cytoplasmiccompartments is undesirable. After harvesting, cells are prepared as asingle-cell suspension; cells are then incubated with an anti-nucleolinantibody usually after blocking non-specific binding sites. Preferably,the anti-nucleolin antibody is labeled with a fluorescent marker. If theantibody is not labeled with a fluorescent marker, a second antibodythat is immunoreactive with the first antibody and contains afluorescent marker is used. After sufficient washing to ensure thatexcess or unbound antibodies are removed, the cells are ready for flowcytometry.

[0087] Biochemical-Based Approaches:

[0088] In these approaches, it is first desirable to isolate plasmamembrane proteins from other cellular compartments. This may be done inany number of ways, such as simple cell extraction, differentialextraction or mechanical disruption followed by separation of cellularcompartments on gradients (such as sucrose or polydextran) bycentrifugation, extraction followed by immunoselecting appropriatecellular compartments with plasma membrane-specific antibodies, etc. Anexample of such an approach is described in Naito et al. (1988) and Yaoet al. (1996b). Extracting reagents are well known. For examples,solvents such as methanol may be occasionally useful. More likely,detergents, such as t-octylphenoxypolyethoxyethanol (also known aspolyethylene glycol tert-octylphenyl ether) are particularly useful forsimple extractions. Also useful are glucopyranosides, maltopyranosides,maltosides, polyoxyethylene esters, other polyoxyethylene ethers, saltsof alginic, caprylic, cholic 1-decanesulfonic, deoxycholic, dioctylsulfosuccinate, 1-dodecanesulfonic, glyocholic, glycodeoxycholic,1-heptanesulfonic, 1-hexanesulfonic, N-lauroylsacrosine, lauryl sulfate(e.g., SDS), 1-nonanesulfonic, 1-octanesulfonic, 1-pentanesulfonic,taurocholic and tauodexycholic acids; sodium 7-ethyl-2-methyl-4-undecylsulfate, and sodium 2-ethylhexyl sulfate. Other useful detergentsinclude(3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate,(3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane-sulfonate,N-decyl-, N-dodecyl-, N-hexadecyl-, N-octadecyl-,N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonates andphosphatidylcholine. Less useful, but may be helpful in some cases, arealkyltrimethylammonium bromides, benzalkonium chloride, benzethoniumchloride, benzyldimethyldodecylammonium bromide,benzyldimethylhexadecylammonium chloride, cetyldimethylethylammoniumbromide, cetylpyridinium, decamethonium bormide,dimethyldioctadecylammonium bromide, methylbenzethonium chloride,methyltiroctylammonium chloride, andN,N′,N′-polyoxyehtlylene(10)-N-tallow-1,3-diaminopropane. The differentextracting reagents may be used singly or in combination; they may beprepared in simple aqueous solutions or suitable buffers.

[0089] Polyethylene glycol ter-octylphenyl ether is particularly usefulfor differential extraction by taking advantage of the low cloud pointto separate membrane proteins from soluble proteins into two differentphases.

[0090] Extraction buffers may contain protease inhibitors, such asaprotinin, benzamidine, antipain, pepstatin and iodoacetamide.

[0091] Extracts are then assayed for nucleolin expression. For thosetechniques that separate surface plasma membrane from other cellularcomponents (especially the nucleus), the nucleolin detecting agents neednot be specific for extracellular plasma membrane nucleolin epitopes.

[0092] Immunosorbent Assay (ELISA) (Ausubel et al. 1987)

[0093] Various types of enzyme linked immunosorbent assays (ELISAs) todetect protein expression are known, and these are applicable tonucleolin detection. However, other ELISA-like assays includeradio-immunoassays and other non-enzyme linked antibody binding assaysand procedures. In these assays, the cell surface proteins are theprinciple components in the cell preparation.

[0094] The double antibody-sandwich ELISA technique is especiallyuseful. The basic protocol for a double antibody-sandwich ELISA is asfollows: A plate is coated with anti-nucleolin antibodies (captureantibodies). The plate is then washed with a blocking agent, such asBSA, to block non-specific binding of proteins (antibodies or antigens)to the test plate. The test sample is then incubated on the plate coatedwith the capture antibodies. The plate is then washed, incubated withanti-nucleolin antibodies, washed again, and incubated with a specificantibody-labeled conjugates and the signal appropriately detected.

[0095] In other ELISAs, proteins or peptides are immobilized onto aselected surface, the surface exhibit may have affinity for proteins,such as the wells of a specially-treated polystyrene microtiter plate.After washing to remove incompletely adsorbed material, one would thengenerally desire to bind or coat with a nonspecific protein that isknown to be antigenically neutral with anti-nucleolin antibodies, suchas BSA or casein, onto the well bottom. This step allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antibodies ontothe surface. When the antibodies were created in an animal byconjugating a polypeptide to a protein (e.g., BSA), a different proteinis usually used as a blocking agent, because of the possibility of thepresence of antibodies to the blocking protein the antibody composition.

[0096] After binding of nucleolin to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with ananti-nucleolin antibody composition in a manner conducive to immunecomplex (antigen/antibody) formation. Such conditions include dilutingthe antibody composition with diluents such as BSA, bovine γ globulin(BGG) and PBS/Polyoxyethylenesorbitan monolaurate. These added agentsalso assist in the reduction of nonspecific background signal. Thelayered antibody composition is then allowed to incubate for, e.g., from2 to 4 hours at 25° C. to 37° C. Following incubation, the antibodycomposition-contacted surface is washed so as to removenon-immunocomplexed material. One washing procedure includes washingwith a PBS/polyoxyethylenesorbitan monolaurate or borate buffersolution.

[0097] Following formation of specific immunocomplexes between the testsample and the antibody and subsequent washing, immunocomplex formationis detected using a second antibody having specificity for theanti-nucleolin antibody. For detection, the secondary antibody isassociated with detectable label, such as an enzyme or a fluorescentmolecule. A number of immunoassays are discussed in U.S. Pat. Nos.5,736,348, 5,192,660, and 4,474,892.

[0098] Western Blotting (Ausubel et al., 1987)

[0099] Western blotting methods are well known. Generally, a proteinsample is subjected to sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) at such conditions as to yield an appropriateseparation of proteins within the sample. The proteins are thentransferred to a membrane (e.g., nitrocellulose, nylon, etc.) in such away as to maintain the relative positions of the proteins to each other.

[0100] Visibly labeled proteins of known molecular weight may beincluded within a lane of the gel. These proteins serve as a control toinsure adequate transfer of the proteins to the membrane, as well asmolecular weight markers for determining the relative molecular weightof other proteins on the blot. Alternatively, unlabeled marker proteins(or in some rare instances, no marker proteins) are detected aftertransfer with Brilliant Blue (G or R; Sigma; St. Louis, Mo.) otherprotein dyes. After protein transfer, the membrane is submersed in ablocking solution to prevent nonspecific binding of the primaryantibody.

[0101] The primary antibody, e.g., anti-nucleolin, may be labeled andthe presence and molecular weight of the antigen may be determined bydetecting the label at a specific location on the membrane. However, theprimary antibody may not be labeled, and the blot is further reactedwith a labeled second antibody. This secondary antibody isimmunoreactive with the primary antibody; for example, the secondaryantibody may be one to rabbit imunoglobulins and labeled with alkalinephosphatase. An apparatus for and methods of performing Western blotsare described in U.S. Pat. No. 5,567,595.

[0102] Immunoprecipitation (Ausubel et al., 1987: Harlow and Lane, 1999)

[0103] Protein expression can be determined and quantified by isolatingantigens using immunoprecipitation. Methods of immunoprecipitations aredescribed in U.S. Pat. No. 5,629,197. Immunoprecipitation involves theseparation of the target antigen component from a complex mixture and isused to discriminate or isolate minute amounts of protein. For theisolation of cell-surface proteins, nonionic salts are often used.

[0104] For example, an immunoprecipitation from whole cells may beperformed as follows. Cells are extracted with one or more detergents(see above), such as, for example, 1%t-octylphenoxypolyethoxyethanol/0.1% SDS/150 mM NaCl in 20 mM Trisbuffer, pH 8.6. After extraction, which may be aided by agitation,insoluble debris is removed using a centrifuge. Anti-nucleolin antibodyis added to the extracts, and then the samples are incubated 30 minutesto overnight at 4° C. Staphylococcus aureus or recombinantly-producedProtein A or Group C Staphylococcus Protein G conjugated to sepharose ortris-acryl beads are then added. In those instances when theanti-nucleolin antibody does not bind well to Protein A, IgG Abs thatrecognize antibodies of the animal in which the anti-nucleolin antibodywas made is simultaneously added. The samples are then incubated withgentle agitation for around 2 hours at 4° C. The beads or bacterialcells, now bound to the antibody-antigen complexes, are thoroughlywashed, usually first with either the extraction solution or a high saltbuffer, then a salt-less buffer or water to remove nonspecifically-boundproteins and residual detergent molecules. After removing residualbuffer, the beads are incubated with a buffer, such as electrophoresissample buffer, and then subjected to 95° C. for 3-5 minutes to elutebound proteins from the beads. The samples are then ready for analysisand nucleolin detection.

[0105] Other methods:

[0106] Immunoselection Procedures (other than FACS) (Ausubel et al.,1987)

[0107] Cells expressing plasma membrane nucleolin can be easily isolatedby “panning” on plastic plates coated with anti-antibody antibodies(Wysocki and Sato, 1978). Panning has many advantages over otherimmunoselection procedures: It is fast, efficient (10⁷ cells can easilybe panned on two 60-mm plastic plates in 30 minutes), and inexpensive.

[0108] In general, a single cell suspension is labeled with ananti-nucleolin antibody, and then is incubated on a substrate coatedwith a secondary antibody (with nonspecific binding sites blocked).After 1 to 3 hours incubation at room temperature, non-adherent cellsare washed away. In this embodiment, bound cells indicate that nucleolinis expressed in the plasma membrane, indicating a neoplastic cell.

[0109] Detecting Nucleolin: Oligonucleotide-Based Methods

[0110] GROs and other oligonucleotides that recognize and bind nucleolin(Bates et al., 1999; Miller et al., WO 00/61597, 2000; Xu et al., 2001)can be used much the same way as antibodies are. Examples of suitableassays are given below. In some cases, incorporating the GRO nucleotidesinto larger nucleic acid sequences may be advantageous; for example, tofacilitate binding of a GRO nucleic acid to a substrate withoutdenaturing the nucleolin-binding site.

[0111] Useful GROs that bind nucleolin (and also have the biologicalproperty of inhibiting cancer cell growth) have been described (Bates etal., 1999; Miller et al., WO 00/61597, 2000; Xu et al., 2001). Theyinclude those shown in Table 2. Control GROs are useful for detectingbackground signal levels. TABLE 2 Non-antisense GRO that bind nucleolinand non-binding controls^(1,2,3) SEQ ID GRO Sequence NO: GRO29A¹tttggtggtg gtggttgtgg tggtggtgg 1 GRO29-2 tttggtggtg gtggttttggtggtggtgg 2 GRO29-3 tttggtggtg gtggtggtgg tggtggtgg 3 GRO29-5 tttggtggtggtggtttggg tggtggtgg 4 GRO29-13 tggtggtggt ggt 5 GRO14C ggtggttgtg gtgg6 GRO15A gttgtttggg gtggt 7 GRO15B² ttgggggggg tgggt 8 GRO25A ggttggggtgggtggggtgg gtggg 9 GRO26B¹ ggtggtggtg gttgtggtgg tggtgg 10 GRO28Atttggtggtg gtggttgtgg tggtggtg 11 GRO28B tttggtggtg gtggtgtggt ggtggtgg12 GRO29-6 ggtggtggtg gttgtggtgg tggtggttt 13 GRO32A ggtggttgtggtggttgtgg tggttgtggt gg 14 GRO32B tttggtggtg gtggttgtgg tggtggtggt tt15 GRO56A ggtggtggtg gttgtggtgg tggtggttgt 16 ggtggtggtg gttgtggtggtggtgg GRO tttcctcctc ctccttctcc tcctcctcc 18 GRO A ttagggttagggttagggtt aggg 19 GRO B ggtggtggtg g 20 GRO C ggtggttgtg gtgg 21 GRO Dggttggtgtg gttgg 22 GRO E gggttttggg 23 GRO F ggttttggtt ttggttttgg 24GRO G¹ ggttggtgtg gttgg 25 GRO H¹ ggggttttgg gg 26 GRO I¹ gggttttggg 27GRO J¹ ggggttttgg ggttttgggg ttttgggg 28 GRO K¹ ttggggttgg ggttggggttgggg 29 GRO L¹ gggtgggtgg gtgggt 30 GRO M¹ ggttttggtt ttggttttgg ttttgg31 GRO N² tttcctcctc Ctccttctcc tcctcctcc 32 GRO O² cctcctcctccttctcctcc tcctcc 33 GRO P² tggggt 34 GRO Q² gcatgct 35 GRO R²gcggtttgcg g 36 GRO S² tagg 37 GRO T² ggggttgggg tgtggggttg ggg 38

[0112] Cytological-Based Approaches:

[0113] Cellular Localization/Labeling (Relative of Immuno-BasedLocalization/Labeling Assays)

[0114] The procedures outlined above for the immuno-based localizationassays (such as immunofluorescence or FACS) are also applicable to thoseassays wherein the detecting reagent is a nucleolin-binding GRO.Modifications include those to prevent non-specific binding, usingdenatured DNA, such as from salmon sperm instead of a protein such asBSA. For detection, similar labels as outlined above are also useful aslong as the GRO can be derivatized with the label in some form. For thispurpose, biotin-avidin nucleic acid labeling systems are especiallyconvenient, as are digoxigenin ones (Ausubel et al., 1987). Thesynthesis of biotinylated nucleotides has been described (Langer et al.,1981). Biotin, a water-soluble vitamin, can covalently attached to theC5 position of the pyrimidine ring via an alylamine linker arm; biotinnon-covalently binds avidin or streptavidin, which may be easilylabeled. Alternatively, biotin is added to oligonucleotides duringsynthesis by coupling to the 5′-hydroxyl of the terminal nucleotide.Digoxigenin-11-dUTP can be incorporated into DNA by either nicktranslation or random oligonucleotide-primed synthesis protocols.Digoxigenin is detected using labeled anti-digoxigenin antibodies.Convenient digoxigenin systems are commercially available (RocheMolecular Biochemicals; Indianapolis, Ind.). An example of a procedureusing oligonucleotides to detect and localize proteins has beendescribed by Davis et al., 1998.

[0115] Biochemical-Based Approaches:

[0116] GROs may also be used in a similar fashion as antibodies todetect nucleolin in biochemical approaches, as described above. Forexample, “Southwestern”-type blotting experiments may be performed withGROs (Bates et al., 1999; Miller et al., WO 00/61597, 2000). After cellshave been appropriately extracted (for example, differentially toseparate plasma membrane proteins from intracellular proteins), theproteins are subjected to electrophoresis on polyacrylamide gels andtransferred to a substrate, such as a polyvinliden difluoride membrane.Proteins are denatured and renatured by washing for 30 minutes at 4° C.with 6 M gaunidine-HCl, followed by washes in 3 M, 1.5 M and 0.75 Mguanidine HCl in 25 mM HEPES (pH 7.9)/4 mM KCl/3 mM MgCl₂). Afterblocking non-specific binding sites with 5% non-fat dried milk in HEPESbuffer, the labeled GRO is hybridized for 2 hours at 4° C. in HEPESbinding buffer supplemented with 0.25% NDM, 0.05% NP-40, 400 ng/mlsalmon sperm DNA and 100 ng/ml of an unrelated mixed sequenceoligonucleotide, such as tcgagaaaaa ctctcctctc cttccttcct ctcca; SEQ IDNO:17. After washing with HEPES binding buffer, the signal is detectedappropriately.

[0117] Other methods:

[0118] Arrays

[0119] Arrays of Immobilized Nucleolin-Binding Reagents on Chips

[0120] A chip is an array of regions containing immobilized molecules,separated by regions containing no molecules or immobilized molecules ata much lower density. For example, a protein chip may be prepared byapplying nucleolin-binding antibodies; an “aptamer”-like chip may beprepared by applying nucleolin binding GROs. The remaining regions areleft uncovered or are covered with inert molecules. The arrays can berinsed to remove all but the specifically immobilized polypeptides ornucleic acids. In addition, chips may also be prepared containingmultiple nucleolin-binding antibodies (Table 1), nucleic acids (such asGROs; Table 2), or both, and may contain control antibodies and/ornucleic acids that are non-reactive with nucleolin. Such an array wouldallow for simultaneous test confirmation, duplication and internalcontrols.

[0121] Proteins, such as anti-nucleolin antibodies, can be immobilizedonto solid supports by simple chemical reactions, including thecondensation of amines with carboxylic acids and the formation ofdisulfides. This covalent immobilization of proteins on inert substratescan prevent high background signals due to non-specific adsorption.Substrates derivatized with other molecules, such as biotin, are alsouseful when the protein to be immobilized is derivatized with avidin orstreptavidin, or vice-versa. In some rare cases, especially whenanti-nucleolin antibody-encoding nucleic acid sequences are available,fusion polypeptides comprising anti-nucleolin antibody may beadvantageous for immobilization onto a substrate.

[0122] The surface may be any material to which a the nucleolin bindingagent can be immobilized. For example, the surface may be metal, glass,ceramic, polymer, wood or biological tissue. The surface may include asubstrate of a given material and a layer or layers of another materialon a portion or the entire surface of the substrate.

[0123] The surfaces may be any of the common surfaces used for affinitychromatography, such as those used for immobilization of glutathione forthe purification of GST fusion polypeptides. The surfaces for affinitychromatography include, for example, sepharose, agarose, polyacrylamide,polystyrene and dextran. The surface need not be a solid, but may be acolloid, an exfoliated mineral clay, a lipid monolayer, a lipid bilayer,a gel, or a porous material.

[0124] The immobilization method desirably controls the position of thenucleolin binding agent on the surface; for example, enabling theantigen binding portions of antibodies unattached to the substrate,while the non-antigen binding portions are rooted to the substrate. Bycontrolling the position of individual reactant ligands, patterns orarrays of the ligands may be produced. The portions of the surface thatare not occupied by the nucleolin-binding reagent do not allownon-specific adsorption of polypeptides or polynucleotides. In thisembodiment, a sample from a subject, for example, blood, is passed overa chip containing nucleolin-binding molecules. A biosensing device, suchas machine that detects changes in surface plasmon resonance, is thenused to detect bound nucleolin. BIAcore (Uppsala, Sweden) chips serve asexamples of useful chips and detection machines.

[0125] Prognostic Assays

[0126] Diagnostic methods can furthermore be used to identify subjectshaving, or at risk of developing, a neoplasia at an early stage ofdisease development, since the surface expression of nucleolin can bedetected earlier than in conventional methods. Prognostic assays can beused to identify a subject having or at risk for developing a neoplasia,such as a subject who has a family history of harmful neoplasias,especially cancers. A method for identifying such an individual wouldinclude a test sample obtained from a subject and testing for cellsurface localization of nucleolin.

[0127] In another embodiment, detecting plasma membrane nucleolin andthen either qualitatively or quantitatively assessing the amount ofnucleolin (usually indirectly through the signal generated from boundnucleolin molecules) can indicate the rate of cell proliferation, sinceplasma membrane nucleolin levels correlate with cell proliferationrates.

[0128] Kits

[0129] Kits, containers, packs, or dispensers containing nucleolinprobes and detection reagents, together with instructions foradministration, may be assembled. When supplied as a kit, the differentcomponents may be packaged in separate containers and admixedimmediately before use. Such packaging of the components separately maypermit long-term storage without losing the active components'functions.

[0130] Kits may also include reagents in separate containers thatfacilitate the execution of a specific test, such as diagnostic tests.For example, non-nucleolin-binding GROs may be supplied for internalnegative controls, or nucleolin and a nucleolin-binding reagent forinternal positive controls. The components of a kit are ananti-nucleolin agent used to probe for nucleolin, a control sample, andoptionally a composition to detect nucleolin. Examples of anti-nucleolinagents include an anti-nucleolin antibody (e.g., as shown in Table 1) orfragment thereof; if labeled, then a nucleolin-binding detection reagentis superfluous. A nucleolin-binding oligonucleotide (e.g., as shown inTable 2), which may be derivatized such that a second labeled reagentmay bind (such as biotin). However, if a labeled GRO nucleic acid isprovided, then a second labeled reagent is superfluous. Examples ofdetection reagents include: labeled secondary antibodies, for example,an anti-mouse pAb made in donkey and then tagged with a fluorophore suchas rhodamine, or a labeled reagent to detect oligonucleotides such asGROs; for example, avidin or streptavidin linked to horseradishperoxidase when the probe is biotinylated. Control components mayinclude: normal serum from the animal in which a secondary antibody wasmade; a solution containing nucleolin polypeptide or nucleolin bindingoligonucleotide; a dot blot of nucleolin protein to assaynucleolin-binding reagent reactivity; or fixed or preserved cells thatexpress nucleolin in the plasma membrane. Other components may includebuffers, fixatives, blocking solutions, microscope slides and/or coverslips or other suitable substrates for analysis, such as microtiterplates; detergent or detergent solutions or other cell extractionreagents; miscellaneous reagents, protease inhibitors, variouscontainers and miscellaneous tools and equipment to facilitate theassays.

[0131] (a) Containers or Vessels

[0132] The reagents included in the kits can be supplied in containersof any sort such that the life of the different components are preservedand are not adsorbed or altered by the materials of the container. Forexample, sealed glass ampules may contain lyophilized nucleolin bindingreagents (such as anti-nucleolin antibodies or nucleolin-bindingoligonucleotides) or buffers that have been packaged under a neutral,non-reacting gas, such as nitrogen. Ampules may consist of any suitablematerial, such as glass, organic polymers (i.e., polycarbonate,polystyrene, etc.), ceramic, metal or any other material typicallyemployed to hold reagents. Other examples of suitable containers includesimple bottles that may be fabricated from similar substances asampules, and envelopes that may have foil-lined interiors, such asaluminum or alloy. Other containers include test tubes, vials, flasks,bottles, syringes, or the like. Containers may have a sterile accessport, such as a bottle having a stopper that can be pierced by ahypodermic injection needle. Other containers may have two compartmentsthat are separated by a readily removable membrane that upon removalpermits the components to mix. Removable membranes may be glass,plastic, rubber, etc.

[0133] (b) Instructional Materials

[0134] Kits may also be supplied with instructional materials.Instructions may be printed on paper or other substrate and/or may besupplied as an electronic-readable medium, such as a floppy disc,CD-ROM, DVD-ROM, DVD, videotape, audio tape, etc. Detailed instructionsmay not be physically associated with the kit; instead, a user may bedirected to an internet web site specified by the manufacturer ordistributor of the kit, or supplied as electronic mail.

[0135] The following examples are intended to illustrate the presentinvention without limitation.

EXAMPLES Example 1 Immunofluorescent Labeling of Plasma MembraneNucleolin in Cells

[0136] This example illustrates a procedure that stains nuclearnucleolin, or only plasma membrane nucleolin.

[0137] Cells from the cell lines DU145 (human prostrate cancer),MDA-MB-231 (human breast cancer) HeLa (human cervical cancer) and HS27(normal skin fibroblasts) (all available from ATCC; Manassas, Va.) werereleased from culture substrates with trypsin, resuspended into singlecells and plated onto microscope slides. The slides seeded with cellswere incubated at 37° C. until they were well attached, as assayed byvisual inspection using a microscope. After rinsing the attached cellsonce with PBS for two minutes, they were fixed in 4% formaldehyde/PBSfor at least 15 minutes at 22° C. For nuclear nucleolin staining, cellsare permeabilized with 1% Triton X-100 prior to contacting withantibody. After washing twice with PBS, 5 minutes/wash, non-specificbinding sites were blocked for 15-60 minutes with 1% NGS/PBS at 22° C.,and then incubated with mouse anti-nucleolin antibodies diluted in 1%NGS/PBS or PBS/Tween (0.05%-0.1%) for 1 hour to overnight at 4° C. Thesamples were washed four times, 5 minutes each with PBS, and thenincubated with goat anti-mouse pAb labeled with FITC-labeled secondaryantibodies diluted in PBS for 1 hour at 22° C. After again washing fourtimes with PBS for 5 minutes each, the samples were mounted in Mowiolmounting media (prepared as follows: 9 ml/glycerol and 3.36 g Mowiol40-88 were agitated for 1 h at 22° C. Then, 9 ml of water was thenadded, and agitation continued for 2 h at 22° C. Tris (0.2 M, pH 8.5; 18ml) was then added, and the solution incubated for 6 h at 50° C. untilthe solids were almost completely dissolved. After centrifugation at5,000×g, the liquid phase was used for mounting), observed under amicroscope, and photographed.

[0138]FIGS. 1 and 2 show nuclear (FIG. 1) and plasma membrane (FIG. 2)nucleolin staining in the various cell lines. Shown areimmunofluorescent (FIGS. 1 and 2; panels B, D, F, H) and parallel phasecontrast micrographs (FIGS. 1 and 2; panels A, C, E, G); DU145 cells areshown in A and B; MDA-MB-231 cells are shown in C and D; HeLa cells areshown in E and F; and HS27 cells are shown in G and H. All cell linesshow clear nuclear nucleolin staining (FIGS. 1A, 1C, 1E and 1G). Notethat when the cells are not permeabilized, thus restricting antibodyaccess to the surface plasma membrane, the normal skin cell line, HS27,is completely negative for plasma membrane staining (FIG. 2H) whilecancer cells show strong staining (FIGS. 2B, 2D, 2F and 2H). Stainingplasma membrane nucleolin is thus a superior method for diagnosis andprognosis compared to nuclear nucleolin or silver-staining NORs.

Example 2 Correlation of the Degree of Plasma Membrane NucleolinExpression and Cancer Aggressiveness

[0139] This experiment demonstrates that cell lines with high levels ofplasma membrane nucleolin correspond to those with the most rapidproliferation.

[0140] Two cancer cell lines, DU145 and HeLa, and one normal cell line,HS27, were assayed for proliferation rate and compared. Cell doublingtime is calculated by determining cell density at regular intervalsusing the MTT assay (based upon the ability of living cells to reduce3-(van de Loosdrecht et al., 1994)-2,5 diphenyltetrazolium bromide (MTT)into formazan; (van de Loosdrecht et al., 1994)), and confirmed bycounting the cells using trypan blue exclusion.

[0141]FIG. 3 shows the comparative proliferation rates of DU145(squares), HeLa (diamonds) and HS27 (circles) as measured by MTT assay.Until 3 days of culture, growth rates are similar, but after 3 days,HeLa and DU145 increase at a faster rate than the normal HS27 cells.Although MDA-MB-231 was not included in this experiment, proliferationrate has been determined to be DU145>MDA-MB-231>HeLa >HS27. Note thatthe cell lines with high levels of plasma membrane nucleolin (see FIG.2) correspond to those with the most rapid proliferation (DU145 andHeLa).

Example 3 Immunofluorescent Labeling of Nucleolin in Paraffin-EmbeddedTissue Sections

[0142] This example provides a suitable technique to detect and localizenucleolin in a fixed sample that has been embedded.

[0143] Sections of cells fixed and embedded in paraffin wax and anchoredon microscope slides were washed in three changes of xylene (2 minuteseach) to remove the paraffin, hydrated in graded alcohols (series 100%,95% and 70%; 2 minutes each), and placed in PBS for 5 minutes. Antigenrecovery used the approach of low temperature antigen retrieval (LTAR;(Shi et al., 1997; Shi et al., 2001)): After digestion with 0.1%trypsin-EDTA (v/v) (Invitrogen Corp.; Carlsbad, Calif.) diluted in PBSfor 15 minutes at 37° C./5% CO₂, the samples were washed with deionizedwater and incubated in 10 mM citrate buffer (pH 6) for 2 hours at 80° C.After cooling, the slides were rinsed with deionized water and then PBS.

[0144] Non-specific binding sites were blocked by incubation in 3% BSAin PBS for 30 minutes at 22° C. The samples were then incubated with 4μg/ml mouse anti-nucleolin mAb (Santa Cruz) diluted in PBS/1% NGS at 4°C. overnight. The samples were then brought to 22° C., washed four timeswith PBS for 5 minutes, and then reacted with 50 μg/mlAlexa488-conjugated goat anti-mouse IgG (Molecular Probes; Eugene,Oreg.) and 2 μg/ml propidium iodide diluted in PBS/1% NGS for 1 hour at22° C. After washing four times with PBS for 5 minutes, the samples weremounted in Mowiol mounting medium and observed under a fluorescentmicroscope.

[0145]FIG. 4 shows the results of such an experiment. A clinical sampleof a squamous cell carcinoma of the head and neck was prepared andprobed for plasma membrane nucleolin. Plasma membrane nucleolin signalwas relegated to malignant, neoplastic cells. FIG. 4A shows theimmunofluorescent signal obtained from probing for nucleolin; the nucleiare counterstained with a DNA-intercalating dye. FIG. 4B shows aparallel phase contrast micrograph. FIGS. 4C and 4D are duplicates ofFIGS. 4A and 4B, except markings have been added to better indicateareas of staining. In region 1, the signal is strong on the cells (faintsignal in relation to the nuclear staining in FIG. 4A); these cells arein loosely-organized tissue and are less densely-packed, suggesting thatthey are malignant. In region 2, normal cells (as delineated bywell-packed cells and organized tissue), cells display no plasmamembrane nucleolin signal.

Example 4 Plasma Membrane Nucleolin Expression in Lung Carcinoma Cells

[0146] This example demonstrates that lung carcinoma cells can be easilyidentified by staining for plasma membrane nucleolin.

[0147] NCI-H1299 (non-small cell lung carcinoma isolated from H. sapienslymph node; (Giaccone et al., 1992; Lin and Chang, 1996)) and NCI-H82(small cell lung carcinoma cells, H. sapiens, (Carney et al., 1985;Little et al., 1983; Takahashi et al., 1989)) cells were released fromculture substrates with trypsin, resuspended into single cells andplated onto microscope slides. The cells were incubated at 37° C. untilthey were well-attached as assayed by visual inspection using amicroscope. After rinsing the cells once with PBS for 2 minutes, theywere fixed in 4% formaldehyde/PBS for at least 15 minutes at 22° C.After washing twice with PBS, 5 minutes/wash, non-specific binding siteswere blocked for 15-60 minutes with 1% NGS/PBS at 22° C., and thenincubated with mouse anti-nucleolin antibodies for 1 hour to overnightat 4° C. The samples were washed four times, 5 minutes each with PBS andthen incubated with goat anti-mouse pAb labeled with FITC-labeledsecondary antibodies diluted in PBS with propidium iodide (to stainnuclei) for 1 hour at 22° C. After again washing four times with PBS for5 minutes each, the samples were mounted in Mowiol mounting media,observed under a microscope and photographed.

[0148]FIG. 5 shows whole cells probed for plasma membrane nucleolin ofthe two lung cancer cell lines, NCI-H82 (FIG. 5A; a parallel phasecontrast image is shown in 5B) and NCI-H1299 (FIG. 5C; a parallel phasecontrast image is shown in 5D).

[0149] In both cell lines, plasma membrane nucleolin staining is strong;examples of well-stained cells are denoted by asterisk (*) in FIGS. 5Aand 5C.

Example 5 Plasma Membrane Nucleolin Staining of Clinical Specimens

[0150] To test the feasibility of using this novel method of assayingplasma membrane nucleolin to diagnose tumor, pre-malignant and malignantcells, clinical specimens from healthy subjects and those suffering froma cancer were collected. Samples from peripheral blood, bone marrow andtumor biopsy samples were obtained and stained for plasma membranenucleolin as described in Example 4. FIG. 6 shows phase contrast (B, D,F) and immunofluorescent images (A, C, E) of peripheral blood (A, B) orbone marrow (C, D and E, F). Highly stained cells for plasma membranenucleolin are marked with an asterisk (*); these were only seen in thosepatients suffering from carcinomas (A, B and C, D), while cells from ahealthy patient did not display any plasma membrane staining (E, F).

Example 6 (Prophetic) Correlation of the Degree of Plasma MembraneNucleolin Expression and Cancer Aggressiveness

[0151] Thirty-three lung carcinoma cell lines are analyzed, mostlyavailable from the American Type Culture Collection (Manassas, Va.).Cell doubling time is calculated by determining cell density at regularintervals using the MTT assay and confirmed by counting the cells usingtrypan blue exclusion. In each experiment HeLa cells (Gey et al., 1952)are included as an internal control. Each value is determined from atleast two independent experiments with triplicate samples. To determinelevels of nuclear and plasma membrane nucleolin, two methods areimplemented. First, nuclear and plasma membrane extracts are preparedfrom each cell line using methods that have as described (Ausubel etal., 1987; Bates et al., 1999; Yao et al., 1996a). Briefly, cells areharvested and resuspended in a hypotonic buffer, then allowed to swellon ice for several minutes. Cells are lysed using a Dounce homogenizer,and nuclei are collected by centrifugation. Nuclei are resuspended in ahigh salt buffer to extract nuclear proteins; salt is then removed bydialysis. Plasma membrane proteins can be isolated from the S-100fraction and are separated from cytosolic proteins and other organellesby centrifugation through a sucrose gradient. Nuclear and PM extractsfrom different cell are analyzed by Western blot analysis (Ausubel etal., 1987) using an anti-nucleolin antidody (Santa Cruz) followed bychemiluminescent visualization. Nucleolin levels are then quantified bydensitometry of the resulting signal recorded on X-ray film andnormalized to the intensity of HeLa extract controls. The secondapproach to determine nucleolin levels involves immunofluorescentprobing of the cell lines for nucleolin. Cells are probed for nucleolinsurface expression in parallel with DU145 cells (Mickey et al., 1977;Stone et al., 1978) as a positive control, HS27 cells as a negativecontrol and HeLa cells as a reference (see FIG. 2). Cells arephotographed and ranked in order of degree of signal, which may also bequantified (using systems that use software and images to quantitatepixels; in this instance, video images are used)or qualitativelyevaluated. The data are then subjected to statistical analysis todemonstrate correlations with the degree of cell proliferation (higherrates of cell proliferation indicate more aggressive cancer cells) withthe intensity of nucleolin signal across the entire sample and withinsubsets.

Example 7 (Prophetic) Lung Cancer Detection

[0152] In this example, patient biopsies, sputum samples and resectedlung tissue are probed for plasma membrane nucleolin, and these resultsare compared to other diagnostic and prognostic markers for lung cancer,utilizing archival and routine clinical specimens for this study.

[0153] Methods

[0154] Specimens including bronchial biopsies, sputum samples, andresected lung tissue are obtained from human subjects, both healthy andthose suffering from lung cancer, and each sample encoded such that atthe time of nucleolin probing and observation, the sample origin isunknown.

[0155] Probing these samples using immunohistochemical techniques arethen implemented. For example, plasma membrane nucleolin is probed withone or more anti-nucleolin Abs selected from Table 1, a signal generatedfrom a flourophore-tagged secondary Ab, and the samples observed andphotographed. Appropriate controls include probing with the secondaryantibody only, probing with no antibodies, probing with pre-immune serumonly, and probing with an antibody known not to react with the celltypes being analyzed. To facilitate visualization and localizationdetermination, the cells can be counterstained with Hoechst 33258 orpropidium iodide (to visualize nuclei) and/or with fluorescent-taggedphalloidin or phallicidin (to visualize the actin cytoskeleton). Thesamples are observed, scored (surface signal indicating plasma membranenucleolin expression) and documented.

References

[0156] Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, et al.1987. Current protocols in molecular biology. John Wiley & Sons, NewYork.

[0157] Babkes, M., A. Ranford, and K. Yaeger. U.S. Pat. No. 6,325,785.2001. Sputum trap manifold with nested caps.

[0158] Bandman, O., H. Yue, N. Corley, and P. Shah. U.S. Pat. No.5,932,475. 1999. Human nucleolin-like protein.

[0159] Bates, P. J., J. B. Kahlon, S. D. Thomas, J. O. Trent, et al.1999. Antiproliferative activity of G-rich oligonucleotides correlateswith protein binding. J Biol Chem. 274:26369-77.

[0160] Byrne, C. J. 1986. Laboratory tests: implications for nursingcare. Addison-Wesley Pub. Co. Health Sciences Division, Menlo Park,Calif. xxi, 756 pp.

[0161] Callebaut, C., J. Blanco, N. Benkirane, B. Krust, et al. 1998.Identification of V3 loop-binding proteins as potential receptorsimplicated in the binding of HIV particles to CD4(+) cells. J Biol Chem.273:21988-97.

[0162] Carney, D. N., A. F. Gazdar, G. Bepler, J. G. Guccion, et al.1985. Establishment and identification of small cell lung cancer celllines having classic and variant features. Cancer Res. 45:2913-23.

[0163] Davis, K. A., Y. Lin, B. Abrams, and S. D. Jayasena. 1998.Staining of cell surface human CD4 with 2′-F-pyrimidine-containing RNAaptamers for flow cytometry. Nucleic Acids Res. 26:3915-24.

[0164] Derenzini, M. 2000. The AgNORs. Micron. 31:117-20.

[0165] Gey, G., W. Coffman, and M. Kubicek. 1952. Tissue culture studiesof the proliferative capacity of cervical carcinoma and normalepithelium. Cancer Res. 12:264.

[0166] Giaccone, G., J. Battey, A. F. Gazdar, H. Oie, et al. 1992.Neuromedin B is present in lung cancer cell lines. Cancer Res.52:2732s-2736s.

[0167] Ginisty, H., H. Sicard, B. Roger, and P. Bouvet. 1999. Structureand functions of nucleolin. J Cell Sci. 112:761-72.

[0168] Goding, J. W. 1996. Monoclonal antibodies: Principles andPractice. Academic Press, San Diego. 492 pp.

[0169] Harlow, E., and D. Lane. 1988. Antibodies: A laboratory manual.Cold Spring Harbor Laboratory Press, Cold Spring Harbor. 726 pp.

[0170] Harlow, E., and D. Lane. 1999. Using antibodies: A laboratorymanual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0171] Kim, C. S., B. B. Berkley, W. M. Abraham, and A. Wanner. 1982. Amicro double capillary method for rheologic measurements of lower airwaysecretions. Bull Eur Physiopathol Respir. 18:915-27.

[0172] King, M., and D. Speert. U.S. Pat. No. 6,339,075. 2002. Use ofdextran and other polysaccharides to improve mucus clearance.

[0173] Langer, P. R., A. A. Waldrop, and D. C. Ward. 1981. Enzymaticsynthesis of biotin-labeled polynucleotides: novel nucleic acid affinityprobes. Proc Natl Acad Sci USA. 78:6633-7.

[0174] Lin, D. L., and C. Chang. 1996. p53 is a mediator forradiation-repressed human TR2 orphan receptor expression in MCF-7 cells,a new pathway from tumor suppressor to member of the steroid receptorsuperfamily. J Biol Chem. 271:14649-52.

[0175] Little, C. D., M. M. Nau, D. N. Carney, A. F. Gazdar, et al.1983. Amplification and expression of the c-myc oncogene in human lungcancer cell lines. Nature. 306:194-6.

[0176] McNicol, A. M., and J. A. Richmond. 1998. Optimizingimmunohistochemistry: antigen retrieval and signal amplification.Histopathology. 32:97-103.

[0177] Mickey, D. D., K. R. Stone, H. Wunderli, G. H. Mickey, et al.1977. Heterotransplantation of a human prostatic adenocarcinoma cellline in nude mice. Cancer Res. 37:4049-58.

[0178] Miller, D., P. Bates, and J. Trent. WO 00/61597. 2000.Antiproliferative activity of G-rich oligonucleotides and method ofusing same to bind to nucleolin.

[0179] Naito, M., H. Hamada, and T. Tsuruo. 1988. ATP/Mg²⁺-dependentbinding of vincristine to the plasma membrane of multidrug-resistantK562 cells. J. Biol Chem. 263:11887-91.

[0180] Orfao, A., and A. Ruiz-Arguelles. 1996. General concepts aboutcell sorting techniques. Clin Biochem. 29:5-9.

[0181] Robinson, J. M., and D. D. Vandre. 2001. Antigen retrieval incells and tissues: enhancement with sodium dodecyl sulfate. HistochemCell Biol. 116:119-30.

[0182] Rubin, B., and M. Newhouse. U.S. Pat. No. 5,925,334. 1999. Use ofsurface active agents to promote mucus clearance.

[0183] Shi, S. R., R. J. Cote, and C. R. Taylor. 1997. Antigen retrievalimmunohistochemistry: past, present, and future. J Histochem Cytochem.45:327-43.

[0184] Shi, S. R., R. J. Cote, and C. R. Taylor. 2001. Antigen retrievaltechniques: current perspectives. J Histochem Cytochem. 49:931-7.

[0185] Sorokina, E. A., and J. G. Kleinman. 1999. Cloning andpreliminary characterization of a calcium-binding protein closelyrelated to nucleolin on the apical surface of inner medullary collectingduct cells. J Biol Chem. 274:27491-6.

[0186] Srivastava, M., and H. B. Pollard. 1999. Molecular dissection ofnucleolin's role in growth and cell proliferation: new insights. FasebJ. 13:1911-22.

[0187] Stone, K. R., D. D. Mickey, H. Wunderli, G. H. Mickey, et al.1978. Isolation of a human prostate carcinoma cell line (DU 145). Int JCancer. 21:274-81.

[0188] Takahashi, T., M. M. Nau, I. Chiba, M. J. Birrer, et al. 1989.p53: a frequent target for genetic abnormalities in lung cancer.Science. 246:491-4.

[0189] Tockman, M. S., J. L. Mulshine, S. Piantadosi, Y. S. Erozan, etal. 1997. Prospective detection of preclinical lung cancer: results fromtwo studies of heterogeneous nuclear ribonucleoprotein A2/B1overexpression. Clin Cancer Res. 3:2237-46.

[0190] Tuteja, R., and N. Tuteja. 1998. Nucleolin: a multifunctionalmajor nucleolar phosphoprotein. Crit Rev Biochem Mol. Biol. 33:407-36.

[0191] van de Loosdrecht, A. A., R. H. Beelen, G. J. Ossenkoppele, M. G.Broekhoven, et al. 1994. A tetrazolium-based calorimetric MTT assay toquantitate human monocyte mediated cytotoxicity against leukemic cellsfrom cell lines and patients with acute myeloid leukemia. J ImmunolMethods. 174:311-20.

[0192] Wysocki, L. J., and V. L. Sato. 1978. “Panning” for lymphocytes:a method for cell selection. Proc Natl Acad Sci USA. 75:2844-8.

[0193] Xu, X., F. Hamhouyia, S. D. Thomas, T. J. Burke, et al. 2001.Inhibition of DNA Replication and Induction of S Phase Cell Cycle Arrestby G-rich Oligonucleotides. J Biol Chem. 276:43221-30.

[0194] Yao, G. Q., S. Corrias, and Y. C. Cheng. 1996a. Identification oftwo oligodeoxyribonucleotide binding proteins on plasma membranes ofhuman cell lines. Biochem Pharmacol. 51:431-6.

[0195] Yao, G. Q., S. Corrias, and Y. C. Cheng. 1996b. Identification oftwo oligodeoxyribonucleotide binding proteins on plasma membranes ofhuman cell lines. Biochem Pharmacol. 51:431-6.

[0196]

1 38 1 29 DNA Artificial sequence synthetic oligonucleotide 1 tttggtggtggtggttgtgg tggtggtgg 29 2 29 DNA Artificial sequence syntheticoligonucleotide 2 tttggtggtg gtggttttgg tggtggtgg 29 3 29 DNA Artificialsequence synthetic oligonucleotide 3 tttggtggtg gtggtggtgg tggtggtgg 294 29 DNA Artificial sequence synthetic oligonucleotide 4 tttggtggtggtggtttggg tggtggtgg 29 5 13 DNA Artificial sequence syntheticoligonucleotide 5 tggtggtggt ggt 13 6 14 DNA Artificial sequencesynthetic oligonucleotide 6 ggtggttgtg gtgg 14 7 15 DNA Artificialsequence synthetic oligonucleotide 7 gttgtttggg gtggt 15 8 15 DNAArtificial sequence synthetic oligonucleotide 8 ttgggggggg tgggt 15 9 25DNA Artificial sequence synthetic oligonucleotide 9 ggttggggtgggtggggtgg gtggg 25 10 26 DNA Artificial sequence syntheticoligonucleotide 10 ggtggtggtg gttgtggtgg tggtgg 26 11 28 DNA Artificialsequence synthetic oligonucleotide 11 tttggtggtg gtggttgtgg tggtggtg 2812 28 DNA Artificial sequence synthetic oligonucleotide 12 tttggtggtggtggtgtggt ggtggtgg 28 13 29 DNA Artificial sequence syntheticoligonucleotide 13 ggtggtggtg gttgtggtgg tggtggttt 29 14 32 DNAArtificial sequence synthetic oligonucleotide 14 ggtggttgtg gtggttgtggtggttgtggt gg 32 15 32 DNA Artificial sequence synthetic oligonucleotide15 tttggtggtg gtggttgtgg tggtggtggt tt 32 16 56 DNA Artificial sequencesynthetic oligonucleotide 16 ggtggtggtg gttgtggtgg tggtggttgt ggtggtggtggttgtggtgg tggtgg 56 17 35 DNA Artificial sequence syntheticoligonucleotide 17 tcgagaaaaa ctctcctctc cttccttcct ctcca 35 18 29 DNAArtificial Sequence synthetic oligonucleotide 18 tttcctcctc ctccttctcctcctcctcc 29 19 24 DNA Artificial Sequence synthetic oligonucleotide 19ttagggttag ggttagggtt aggg 24 20 11 DNA Artificial Sequence syntheticoligonucleotide 20 ggtggtggtg g 11 21 14 DNA Artificial Sequencesynthetic oligonucleotide 21 ggtggttgtg gtgg 14 22 15 DNA ArtificialSequence synthetic oligonucleotide 22 ggttggtgtg gttgg 15 23 10 DNAArtificial Sequence synthetic oligonucleotide 23 gggttttggg 10 24 20 DNAArtificial Sequence synthetic oligonucleotide 24 ggttttggtt ttggttttgg20 25 15 DNA Artificial sequence synthetic oligonucleotide 25 ggttggtgtggttgg 15 26 12 DNA Artificial sequence synthetic oligonucleotide 26ggggttttgg gg 12 27 10 DNA Artificial sequence synthetic oligonucleotide27 gggttttggg 10 28 28 DNA Artificial sequence synthetic oligonucleotide28 ggggttttgg ggttttgggg ttttgggg 28 29 24 DNA artificial sequencesynthetic oligonucleotide 29 ttggggttgg ggttggggtt gggg 24 30 16 DNAartificial sequence synthetic oligonucleotide 30 gggtgggtgg gtgggt 16 3126 DNA artificial sequence synthetic oligonucleotide 31 ggttttggttttggttttgg ttttgg 26 32 29 DNA artificial sequence syntheticoligonucleotide 32 tttcctcctc ctccttctcc tcctcctcc 29 33 26 DNAartificial sequence synthetic oligonucleotide 33 cctcctcctc cttctcctcctcctcc 26 34 6 DNA artificial sequence synthetic oligonucleotide 34tggggt 6 35 7 DNA artificial sequence synthetic oligonucleotide 35gcatgct 7 36 11 DNA Artificial sequence synthetic oligonucleotide 36gcggtttgcg g 11 37 4 DNA artificial sequence synthetic oligonucleotide37 tagg 4 38 23 DNA artificial sequence synthetic oligonucleotide 38ggggttgggg tgtggggttg ggg 23

1. A method of determining a neoplastic state of a cell, comprising:detecting the presence of plasma membrane nucleolin in the cell.
 2. Themethod of claim 1, wherein the cell is mammalian.
 3. The method of claim2, wherein the cell is monkey, dog, cat, rabbit, cow, pig, goat, guineapig, mouse, rat or sheep.
 4. The method of claim 2, wherein the cell ishuman.
 5. The method of claim 1, wherein the cell is lysed.
 6. Themethod of claim 1 wherein the detecting comprises detectingnucleolin-anti-nucleolin agent complex.
 7. The method of claim 6,wherein the anti-nucleolin agent is a monoclonal antibody thatspecifically binds nucleolin.
 8. The method of claim 7, wherein theantibody is p7-1A4 mouse monoclonal antibody, sc-8031, sc-9893, sc-9892,4E2 or 3G4B2.
 9. The method of claim 6, wherein the anti-nucleolin agentis a nucleolin-binding oligonucleotide.
 10. The method of claim 9,wherein the oligonucleotide is a guanosine-rich oligonucleotide.
 11. Themethod of claim 10, wherein the oligonucleotide comprises a sequenceselected from the group consisting of SEQ ID NOs:1-7; 9-17; 19-30 or 31.12. The method of claim 6, wherein the complex is detected by detectingfluorescence, an enzyme, or radioactivity.
 13. A method of determining aneoplastic state of a cell, comprising quantifying the amount of plasmamembrane nucleolin in the cell.
 14. The method of claim 13, wherein thecell is mammalian.
 15. The method of claim 14, wherein the cell ismonkey, dog, cat, rabbit, cow, pig, goat, guinea pig, mouse, rat orsheep.
 16. The method of claim 15, wherein the cell is human.
 17. Themethod of claim 13, wherein the cell is lysed.
 18. The method of claim13 wherein the quantifying comprises quantifyingnucleolin-anti-nucleolin agent complex.
 19. The method of claim 18,wherein the anti-nucleolin agent is a monoclonal antibody.
 20. Themethod of claim 18, wherein the antibody is p7-1A4 mouse monoclonalantibody, sc-8031, sc-9893, sc-9892, 4E2 or 3G4B2.
 21. The method ofclaim 18, wherein the anti-nucleolin agent is nucleolin-bindingoligonucleotide.
 22. The method of claim 21, wherein the oligonucleotideis a guanosine-rich oligonucleotide.
 23. The method of claim 22, whereinthe oligonucleotide comprises a sequence selected from the groupconsisting of SEQ ID NOs:1-7; 9-17; 19-30 or
 31. 24. The method of claim23, wherein the complex is quantified by quantifying fluorescence, anenzyme, or radioactivity.
 25. A kit for determining a neoplastic stateof a cell, comprising: an anti-nucleolin agent and a control sample. 26.The kit of claim 25, further comprising a second agent that detects anucleolin-anti-nucleolin complex.
 27. The kit of claim 26, wherein theanti-nucleolin agent is an anti-nucleolin antibody, and the second agentis a labeled secondary antibody.
 28. The kit of claim 27, wherein thelabel is at least one enzyme, fluorophore, radioisotope or goldparticle.
 29. The kit of claim 28, wherein the anti-nucleolin agentcomprises biotin, and the second agent comprises avidin or streptavidin.30. The kit of claim 28, wherein the anti-nucleolin agent comprisesdigoxigenin, and the second agent comprises a labeled anti-digoxigeninantibody. 31 The kit of claim 25, further comprising at least one memberselected from the group consisting of a fixative, a buffer, plasticware,serum, serum albumin, non-fat milk, membranes and instructions.
 32. Thekit of claim 25, wherein the anti-nucleolin agentt is labeled.
 33. Thekit of claim 32, wherein the label is at least one enzyme, fluorophore,radioisotope or gold particle.
 34. A method of detecting small lung cellcarcinoma in a subject, comprising: collecting a sample from the subjectcontaining lung cells; and detecting the presence of nucleolin on thesurface of the cells.
 35. The method of claim 34, wherein the sample issputum.
 36. The method of claim 34, wherein the subject is human.
 37. Amethod of diagnosing pre-malignant or malignant cells, comprising:collecting a sample from a subject comprising lung cells; sending thesample to a testing center; determining the neoplastic state of thecells by the method of claim
 1. 38. The method of claim 38, wherein thesubject is human.
 39. The method of claim 39, wherein the sample issputum.
 40. The method of claim 39, wherein the diagnosing is diagnosinglung small cell carcinoma.